The present invention relates to a biosynthetic gene cluster of Micromonospora echinospora spp. calichensis. In particular, the calicheamicin biosynthetic gene cluster contains genes coding for proteins and enzymes used in the biosynthetic pathway and construction of calicheamicin""s aryltetrasaccharide and aglycone, and the gene conferring calicheamicin resistance. The present invention also relates to isolated genes of the biosynthetic cluster and their corresponding proteins. In addition, the invention relates to DNA hybridizing with the calicheamicin gene cluster and the isolated genes of that cluster. The invention also relates to expression vectors containing the biosynthetic gene cluster, the individual genes, or functional variants thereof.
The enediyne antibiotics, which were discovered in the 1980""s, have long been appreciated for their novel molecular architecture, their remarkable biological activity, and their fascinating mode of action. Enediyne antibiotics were originally derived by fermentation of microorganisms, including Micromonospora, Actinomadura, and Streptomyces. Rothstein, D. M., Enediyne Antibiotics as Antitumor Agents, p. 2 (1995). As a class, the enediyne antibiotics have been referred to as the most potent and highly active antitumor reagents yet discovered. Rothstein, D. M., Enediyne Antibiotics as Antitumor Agents, preface (1995).
To date, at least twelve members of this family of antibiotics have been discovered, all of which fall roughly into two categories. The members of the first category of enediynes are classified as chromoprotein enediynes because they possess a novel 9-membered ring chromophore core structure, which also requires a specific associated protein for chromophore stabilization. The members of the second category of enediyne are classified as non-chromoprotein enediynes. These enediynes contain a 10-membered ring, which requires no additional stabilization factors. This enediyne ring structure is often referred to as the xe2x80x9cwarhead.xe2x80x9d The warhead induces DNA damage, which is frequently a double-stranded cleavage and appears to be irreparable. This type of DNA damage is usually nonrepairable for the cell and is most often lethal. Because of these remarkable chemical and biological properties, there has been an intense effort by both the pharmaceutical industry and academia to study these substances with the goal of developing new and clinically useful therapeutic anti-tumor agents.
The 9-membered ring chromoprotein enediyne subfamily is comprised of: neocarzinostatin from Streptomyces carzinostaticus, (Myers, A. G., et al., J. Am. Chem. Soc., 110, 7212-7214 (1988)); kedarcidin from Actinomycete L585-6, (Leet, J. E., et al., J. Am. Chem. Soc., 114, 7946-7948 (1992)), N1999A2 from Streptomyces globisporus, (Yoshida, K., et al. Tetrahedron Lett., 34, 2637-2640 (1993)), maduropeptin from Actinomadura madurea, (Schroeder, D. R., et al., J. Am. Chem. Soc., 116, 9351-9352 (1994)); N1999A2 from Streptomyces sp. AJ9493, (Schroeder, D. R., et al., J. Am. Chem. Soc., 116, 9351-9352 (1994)); actinoxanthin from Actinomyces globisporus, (Khokhlov, A. S., et al., J. Antibiot., XXII, 541-544 (1969)); largomycin from Streptomyces pluricolorescens, (Yamaguchi, T., et al., J. Antibiot., XXIII, 369-372 (1970)); auromomycin from Streptomyces macromomyceticus, (Yamashita, T., et al., J. Antibiot., XXXII, 330-339 (1979)), and sporamycin from Streptosporangium pseudovulgare, (Komiyama, K, et al., J. Antibiot., XXX, 202-208 (1977)), all of which are believed to possess a novel bicylo[7.3.0.]dodecadiynene chromophore core structure essential for biological activity. In addition, with the exception of N1999A2, a required apoprotein acts as a stabilizer and specific carrier for the unstable chromophore, and for its transport and interaction with target DNA.
The non-chromophore enediyne subfamily is comprised of calicheamicin from Micromonospora echinospora spp. calichensis; namenamicin from Polysyncraton lithostrotum; esperamicin from Actinomadura verrucosospora; and dynemicin from Micromonospora chersina. 
Enediyne antibiotics have potential as anticancer agents because of their ability to cleave DNA; however, many of these compounds are too toxic to be used currently in clinical studies. Today, only calicheamicin is known to be currently used in clinical trials; and it has provided promising results as an anticancer agent. For example, MyloTarg(trademark), a calicheamicin-antibody conjugate also known as CMA-676 was approved by the FDA in January of 2000 to treat acute myelogenous leukemia. The enediynes also potentially have utility as anti-infective agents, provided that toxicity can be managed.
Calicheamicin has two distinct structural regions: the aryltetrasaccharide and the aglycone (also known as the warhead). The aryltetrasaccharide displays a highly unusual series of glycosidic, thioester, and hydroxylamine linkages and serves to deliver the drug primarily to specific tracts (5xe2x80x2-TCCT-3xe2x80x2 and 5xe2x80x2-TTTT-3xe2x80x2) within the minor groove of DNA when those sequences are available. However, specificity is also context-dependent. The aglycone of calicheamicin consists of a highly functionalized bicyclo[7.3.1]tridecadiynene core structure with an allylic trisulfide serving as the triggering mechanism. McGahren, W. J.,et al., Enediyne Antibiotics as Antitumor Agents, pp. 75-86 (1995). Once the aryltetrasaccharide is firmly docked, aromatization of the bicyclo[7.3.1]tridecadiynene core structure, via a 1,4-dehydrobenzene-diradical, results in the site specific oxidative double strand scission of the targeted DNA. Zein, N., et al., Science, 240, 1198-1201 (1988). The aglycone undergoes a reaction that yields carbon-centered diradicals, which are responsible for DNA cleavage.
This activity of calicheamicin has sparked considerable interest in the pharmaceutical industry culminating in the recent FDA approval of the calicheamicin-antibody conjugate MyloTarg(trademark) (CMA-676) to treat acute myelogenous leukemia (AML). Additionally, similar strategies have been used in phase I trials to treat breast cancer. A massive program to examine calicheamicin conjugated to alternative delivery systems has also recently been undertaken. Hamann, P. R., et al., 87th Annual Meeting of the American Association of Cancer Research, Washington, D.C., pp. 471 (1996); Hinman, L. M., et al., Cancer Res., 53, 3336 (1993); Hinman, L. M., et al., Enediyne Antibiotics as Antitumor Agents, pp. 87-105 (1995); Sievers, E. L., et al., Blood, 93, 3678-3684 (1999); Siegel, M. M., et al., Anal. Chem., 69, 2716-2726 (1997); Ellestad, G. personal communication.
The biological activity and molecular architecture of calicheamicin has also prompted a search for potentially useful analogs. Of the numerous laboratories producing synthetic analogs, one group has produced a novel calicheamicin xcex311 shown to effectively suppress growth and dissemination of liver metastases in a syngeneic model of murine neuroblastoma. Lode, H. N., et al., Cancer Res., 58, 2925-2928 (1998); Wrasidlo, W., et al., Acta Oncologica, 34, 157-164 (1995). In addition to synthesizing calicheamicin analogs, random mutagenesis of M. echinospora and screening for mutant strains with improved biosynthetic potential has also been pursued. Rothstein, D. M., Enediyne Antibiotics as Antitumor Agents, pp. 107-126 (1995).
The first total synthesis of calicheamicin was reported by Nicolaou and coworkers in 1992. Synthesizing this complex antibiotic, though, presents many disadvantages. For example, Nacelle""s procedure only provides approximately a 0.007% yield and requires 47 steps. Halcomb, R. L., Enediyne Antibiotics as Antitumor Agents, pp. 383-439 (1995). Thus, the total synthesis of calicheamicin remains secondary to the isolation of calicheamicin from large fermentations of M. echinospora. Therefore, methods to produce mass amounts of calicheamicin and potentially useful variants are still needed. Fantini, A., et al., Enediyne Antibiotics as Antitumor Agents, pp. 29-48 (1995). Transferring calicheamicin DNA into producing strains of bacteria, such as Streptomyces, Micromonospora, other actinomyces species, or E. coli, as non-limiting examples, would address this need. However, prior to the discoveries of the present inventors, no cloned M. echinospora genes were available, and only a set of limited studies upon putative M. echinospora promoters were available. Lin, L. S., et al., J. Gen. Microbiol., 138, 1881-1885 (1992); Lin, L. S., et al., J. Bacteriol., 174, 3111-3117 (1992); Baum, E. Z., et al., J. Bacteriol., 171, 6503-6510 (1989); Baum. E. Z., et al., J. Bacteriol., 170, 71-77 (1988).
Calicheamicin""s molecular architecture in conjunction with its useful biological activity and potential therapeutic value brand calicheamicin a target for the study of natural product biosynthesis. While the radical-based mechanism of oxidative DNA cleavage by calicheamicin (i.e. aromatization of the bicyclo[7.3.1]tridecadiynene core structure, via a 1,4-dehydrobenzene-diradical, resulting in the site specific oxidative double strand DNA cleavage) is well understood, it was unknown, prior to this invention, how Micromonospora constructs calicheamicin. As a result, before the present invention, there was a need to discover and understand calicheamicin biosynthesis. Prior to this discovery of the present inventors, knowledge of genes coding for nonchromoprotein enediyne biosynthesis was completely lacking.
The toxicity of the enediyne compounds, including calicheamicin, centers on the problem of directing the compound to cleave only the DNA of interest, such as tumor cell DNA, and not the DNA of the host. Due to calicheamicin""s powerful ability to cleave DNA, scientists have investigated the mechanism by which calicheamicin-producing organism protects itself against the DNA-cleaving activity of the molecule (Rothstein, D. M., Enediyne Antibiotics as Antitumor Agents, p. 77 (1995)). Prior to this invention, knowledge of genes coding for non-chromoprotein enediyne self resistance was completely lacking.
The present invention relates to the first identification, isolation, and cloning of a nonchromoprotein enediyne biosynthetic gene cluster and mapping and nucleotide sequence analysis of the genes within the cluster. The invention provides the entire calicheamicin-biosynthetic cluster and biochemical studies of aryltetrasaccharide biosynthesis. Furthermore, the calicheamicin self-resistance gene and protein have been isolated, as have the genes and resulting enzymes for steps within the calicheainicin cascade. The invention also provides for construction of enediyne overproducing strains, for rational biosynthetic modification of bioactive secondary metabolites, for new drug leads, and for an enediyne combinatorial biosynthesis program.
The present invention provides an isolated nucleic acid molecule from a nonchromoprotein enediyne biosynthetic gene cluster from Micromonospora echinospora comprising said nucleic acid molecule, a portion or portions of said nucleic acid molecule wherein said portion or portions encode a protein, a portion or portions of said nucleic acid molecule wherein said portion or portions encode a biologically active fragment of a protein. The isolated nucleic acid molecule may be single- or double-stranded. As used herein, a nucleic acid molecule, polypeptide, or protein described as being xe2x80x9cfromxe2x80x9d e.g., an organism or gene cluster, may have been isolated from such organism or gene cluster; alternatively, it may be a molecule which has been produced using synthetic, chemical, recombinant, or other such methods and comprise an amino acid or nucleotide sequence which may be isolated from such organism or gene cluster.
The present invention provides forty-eight genes, twenty-seven of which encode structural genes with the remainder encoding a variety of functions. The present invention is drawn to the following genes or nucleic acids: calC (SEQ ID No. 1), calH (SEQ ID No. 3), calG (SEQ ID No. 5), calA (SEQ ID No. 7), calB (SEQ ID No. 9), calD (SEQ ID No. 11, calF (SEQ ID No. 13), calI (SEQ ID No. 15), calJ (SEQ ID No. 17), calK (SEQ ID No. 19), calL (SEQ ID No. 21), calM (SEQ ID No. 23), calN (SEQ ID No. 25), calO (SEQ ID No. 27), calP (SEQ ID No. 29), calQ (SEQ ID No. 31), calR (SEQ ID No. 33), calS (SEQ ID No. 35), calT (SEQ ID No. 37), calU (SEQ ID No. 39), calV (SEQ ID No. 41), calW (SEQ ID No. 43), calX (SEQ ID No. 45), 6MSAS (SEQ ID No. 47), ActI (SEQ ID No. 49), ActII (SEQ ID No. 51), ActIII (SEQ ID No. 53), orf1 (SEQ ID No. 55), orf2 (SEQ ID No. 57), orf3 (SEQ ID No. 59), orf4 (SEQ ID No. 61), orf5 (SEQ ID No. 63), orf6 (SEQ ID No. 65), orf7 (SEQ ID No. 67), orf8 (SEQ ID No. 69), orfI (SEQ ID No. 71), orfII (SEQ ID No. 73), orfIII (SEQ ID No. 75), orfIV (SEQ ID No. 77), orfV (SEQ ID No. 79):, orfVI (SEQ ID No. 81), orfVII (SEQ ID No. 83), orfVIII (SEQ ID No. 85), orfIX (SEQ ID No. 87), orfX (SEQ ID No. 89), orfXI (SEQ ID No. 91), IS-element (DNA) (SEQ ID No. 93), calE (SEQ ID No. 94). The invention is also drawn to the following proteins or putative proteins: CalC (SEQ ID No. 2), CalH (SEQ ID No. 4), CalG (SEQ ID No. 6), CalA (SEQ ID No. 8), CalB (SEQ ID No. 10), CalD (SEQ ID No. 12), CalF (SEQ ID No. 14), CalI (SEQ ID No. 16), CalJ (SEQ ID No. 18), CalK (SEQ ID No. 20), CalL (SEQ ID No. 22), CalM (SEQ ID No. 24), CalN (SEQ ID No. 26), CalO (SEQ ID No. 28), CalP (SEQ ID No. 30), CalQ (SEQ ID No. 32), CalR (SEQ ID No. 34), CalS (SEQ ID No. 36), CalT (SEQ ID No. 38), CalU (SEQ ID No. 40), CalV (SEQ ID No. 42), CalW (SEQ ID No. 44), CalX (SEQ ID No. 46), 6MSAS (SEQ ID No. 48), ActI (SEQ ID No. 50), ActII (SEQ ID No. 52), ActIII (SEQ ID No. 54), Orf1 (SEQ ID No. 56), Orf2 (SEQ ID No. 58), Orf3 (SEQ ID No. 60):, Orf4 SEQ ID No. 62), Orf5 (SEQ ID No. 64), Orf6 (SEQ ID No. 66), Orf7 (SEQ ID No. 68), Orf8 (SEQ ID No. 70), OrfI (SEQ ID No. 72), OrfII (SEQ ID No. 74), OrfIII (SEQ ID No. 76), OrfIV (SEQ ID No. 78), OrfV (SEQ ID No. 80), OrfVI (SEQ ID No. 82), OrfVII (SEQ ID No. 84), OrfIII (SEQ ID No. 86), OrfIX (SEQ ID No. 88), OrfX (SEQ ID No. 90), OrfXI (SEQ ID No. 92), CalE (SEQ ID No. 95).
In one aspect, the present invention is directed to an isolated nucleotide molecule, wherein the nucleotide molecule hybridizes with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94, or a functional derivative of the isolated nucleotide molecule which hybridizes with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94. In one embodiment of the invention, the isolated nucleotide molecule has the nucleotide sequence of at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94, i.e., 100% complementarity (sequence identity) with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94. In another embodiment of the invention, the isolated nucleotide molecule has at least 90% complementarity (sequence identity) with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94. In yet another embodiment of the invention, the isolated nucleotide molecule has at least 80% complementarity (sequence identity) with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94. In yet another embodiment of the invention, the isolated nucleotide molecule has at least 70% complementarity (sequence identity) with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94. In yet another embodiment of the invention, the isolated nucleotide molecule has at least 60% complementarity (sequence identity) with at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94. In still yet another embodiment of the invention, the isolated nucleotide molecule is substantially complementary to at least one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 94.
In another embodiment of the invention, there is provided an isolated protein encoded by a DNA molecule as described herein above, or a functional derivative thereof. A preferred protein has the amino acid sequence of at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, or 95 or a functional variant or derivative of one or more of those polypeptides.
In another embodiment, the present invention provides an isolated nucleic acid molecule from Micromonospora echinospora comprising a nonchromoprotein enediyne biosynthetic gene cluster, a portion or portions of said gene cluster wherein said portion or portions encode a protein, a portion or portions of said gene cluster wherein said portion or portions encode a biologically active fragment of a protein, a single-stranded nucleic acid molecule derived from said gene cluster, or a single-stranded nucleic acid molecule derived from a portion or portions of said gene cluster.
In particular, the present invention provides an isolated nucleic acid molecule from Micromonospora echinospora spp. calichensis that is involved in the biosynthesis of calicheamicin. In another embodiment, the present invention also relates to nucleic acids capable of hybridizing with one or more isolated nucleic acids from a nonchromoprotein enediyne biosynthetic gene cluster from Micromonospora echinospora spp. calichensis. In a further embodiment, the invention provides an expression vector comprising an isolated nucleic acid molecule from a nonchromoprotein enediyne biosynthetic gene cluster from Micromonospora echinospora. In yet a further embodiment the invention provides a cosmid comprising an isolated nucleic acid molecule from a nonchromoprotein enediyne biosynthetic gene cluster from Micromonospora echinospora. 
In preferred embodiments, the invention provides the isolated nucleic acid molecules of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 and 94.
In an additional embodiment, the present invention provides a host cell transformed with an isolated nucleic acid molecule from a nonchromoprotein enediyne biosynthetic gene cluster from Micromonospora echinospora. Host cells can optionally be of bacterial, yeast, fungal, insect, plant or mammalian origin and can be transformed according to standard methods. In a preferred embodiment, the host cell is the bacterium E. coli, Streptomyces spp., or Micromonospora spp. In a more preferred embodiment, the host cell is the bacterium from the genus Streptomyces or from the genus Micromonospora.
In a further embodiment, the invention is directed to a host cell transformed with an expression vector comprising at least one of the nucleotide sequences of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 94 or a portion of portions thereof or an allele or alleles thereof. In preferred embodiments, the host cells produce a biologically functional protein or portion of a protein, which protein or portion thereof is encoded by the expression vector.
In a specific embodiment, the invention is directed to a host cell transformed with an expression vector comprising calC, or a portion(s) or allele(s) thereof, operably linked to regulatory sequences that enable expression of CalC. In another specific embodiment, the invention provides a host cell transformed with an expression vector comprising calH, or a portion(s) or allele(s) thereof, operably linked to regulatory sequences that enable expression of CalH. In a yet further specific embodiment, the invention provides a host cell transformed with an expression vector comprising calQ, or a portion(s) or allele(s) thereof, operably linked to regulatory sequences that enable expression of CalQ. Likewise, the invention provides a host cell transformed with an expression vector comprising calG, or a portion(s) or allele(s) thereof, operably linked to regulatory sequences that enable expression of CalG.
In a yet further embodiment, the invention is directed to a host cell transformed with an expression vector encoding at least one polypeptide comprising the amino acid sequence of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, or 95 or a functional variant of one or more of those polypeptides. In preferred embodiments, the host cells produce a biologically functional protein or portion of a protein, which protein or portion thereof is encoded by the expression vector.
In a specific embodiment, the invention is directed to a host cell transformed with an expression vector encoding CalC, or a functional derivative thereof, operably linked to regulatory sequences that enable expression the encoded polypeptide. In another specific embodiment, the invention provides a host cell transformed with an expression vector encoding CalH, or a functional derivative thereof, operably linked to regulatory sequences that enable expression of the encoded polypeptide. In a yet another specific embodiment, the invention provides a host cell transformed with an expression vector encoding CalQ, or a functional derivative thereof, operably linked to regulatory sequences that enable expression of the encoded polypeptide. Likewise, the invention provides a host cell transformed with an expression vector encoding the CalG, or a functional derivative thereof, operably linked to regulatory sequences that enable expression of the encoded polypeptide.
The invention further provides a method of expressing a protein by culturing a host cell transformed with an expression vector of the present invention, and incubating the host cell for a time and under conditions allowing for protein expression.
In yet another embodiment the invention provides a method of purifying calicheamicin using affinity chromatography. A sample containing calicheamicin is contacted with an affinity matrix having the protein CalC bound thereto, for a time and under conditions allowing calicheamicin to bind to the matrix, eluting calicheamicin from the matrix, and recovering calicheamicin.
In a further embodiment the present invention provides polypeptides comprising the amino acid sequences of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 95.
In yet a further embodiment the invention provides the production of the following two new macrolides: 
The invention further provides a method of conferring calicheamicin resistance to a subject comprising obtaining cells from the subject, transforming the cells with the calicheamicin self-resistance gene, and returning the cells to the subject. Alternatively, the calicheamicin self-resistance gene can be targeted and delivered to the desired host cells through known gene therapy delivery systems.
The invention further provides a method of producing calicheamicin analogs by altering calicheamicin or its bioactive metabolites through the modulation of the expression of calD, E, F, G, H, J, K, N, O, P, Q, S, T U, V, W, X, 6MSAS, actI-III, orfI, orfIII, orfV, and orfVII. Such modulation can be achieved through selective xe2x80x9cknock outxe2x80x9d, as well as heterologous expression of these genes and their products. Various combinations of these either mutated or wild type gene products may be used in either in vitro or in vivo calicheamicin analog production.
The invention further provides a method for increasing the production of calicheamicin through the introduction of multiple copies of positive regulators and transporters and or by eliminating or reducing the expression of negative regulators (e.g., CalA, B, I, L, Orf8). Additionally, upregulation of calicheamicin resistance genes calC, calN and orfXI can be used to decrease the toxicity of calicheamicin to healthy tissues and cells during therapy.
In a yet further embodiment, the invention provides for a method of transposon mediated mutagenesis or moving chromosomal DNA fragments in vivo through expression of the orf3 integrase and the IS insertional element.
The advantages of the present invention are numerous. Isolation of and the ability to clone calicheamicin DNA opens the door for genetic analysis of calicheamicin biosynthesis, as such analysis requires the ability to obtain large quantities of DNA which codes for calicheamicin biosynthesis. Using the teachings of the present invention, one can study calicheamicin biosynthesis via mutagenesis of M. echinospora. For example, one can isolate and characterize mutants blocked in calicheamicin biosynthesis and then analyze their defective or partial calicheamicin products. Additionally, particular a enzyme or enzymes can be overexpressed or underexpressed after subcloning its gene into a host such as E. coli, and the results of such overexpression or underexpression can be studied to reveal the enzyme""s function. Furthermore, the cloning of biosynthetic genes can ultimately result in increased yields of the gene product by cloning and expressing the biosynthetic gene encoding the rate-limiting enzyme back into the producing organism.
Further, it may also be possible to generate novel products by cloning biosynthetic genes into strains that make related compounds. Such genes could endow the host organism with the ability to carry out new reactions on the enediyne nucleus, and thus produce novel drugs. The present invention thus also provides means for biosynthetic modification of bioactive secondary metabolites through enediyne combinatorial biosynthesis. As most pharmaceutical drug leads are inspired by naturally occurring compounds, and given the challenge posed in synthesizing these metabolites, genetic manipulation of the sugar appendage on the metabolites offers avenues for creating potential new drugs. Thus the emerging field of combinatorial biosynthesis has become a rich new source for modified non-natural sugar scaffolds. Marsden, A., et al., Science 1998, 279, 199-201. Problems inherent with the genetic manipulation of the sugar appendage relate to the fact that naturally occurring bioactive secondary metabolites possess unusual carbohydrate ligands, which serve as molecular recognition elements critical for biological activity. Macrolide Antibiotics, Chemistry, Biology and Practice, 1984. Without these essential sugar attachments, the biological activities of most clinically important secondary metabolites are either completely abolished or dramatically decreased. Currently, techniques for the genetic manipulation of the sugar appendage for a given metabolite rely mainly on the alteration and/or deletion of a small subset of genes required to construct and attach each desired sugar moiety. Thus there is a need to develop alternate strategies to construct and attach non-naturally occurring sugars. The present invention addresses this need. The present invention utilizes the fact that glycosyltransferases, which are responsible for the final glycosylation of certain secondary metabolites, show a high degree of promiscuity toward the nucleotide sugar donor. Zhao, L., et al., J. Am. Chem. Soc. 1988, 120, 12159-12160. This unselectivity of the glycosyltransferases has the potential for allowing modification of the crucial glycosylation pattern of natural, or non-natural, secondary metabolite scaffolds in a combinatorial fashion. The present invention discloses a method using the recruitment and collaborative action of sugar genes from a variety of biosynthetic pathways to construct composite gene clusters, which make and attach non-natural sugars.
Insight into how Micromonospora self resistance gene and gene products act to control the toxic effects of calicheanicin offers new avenues of clinical research. For example, knowledge of the mechanisms underlying calicheamicin resistance, as provided by the present disclosure, can provide the means necessary to use higher doses of calicheamicin while simultaneously inhibiting the toxic effects of the drug on non-cancer cells. Additionally, understanding the mechanism behind calicheamicin""s self-resistance may aid in the understanding of self-resistance in other enediyne antibiotics, thereby potentially making useful those enediynes once thought to be too toxic to be viably used as therapeutic agents. The calicheamicin self-resistance mechanisms elucidated utilizing the present invention provide gene therapy approaches, for example, via introduction of enediynes resistance genes into bone marrow cells, thereby increasing resistance and allowing tolerance to chemotherapeutic doses of calicheamicin. Banerjee, D., et al., Stem Cells, 12, 378-385 (1994). Thus, understanding calicheamicin self-resistance will significantly aid continuing clinical studies involving calicheamicin and the enediynes. The present invention addresses this need as it provides for the isolation and characterization of a resistance gene and its associated protein for any nonchromoprotein enediynes.